The objective of this proposal is to understand what role inactivation of the TGF-beta receptor complex plays in the multi-stage development of oral cancers. As the functional unit which transduces the inhibitory signal directed by TGF-beta, this heteromeric receptor complex is composed to two serine/threonine kinases (TbetaR-II and TbetaR-I). Working in obligatory cooperation, TbetaR-II and TbetaR-I indirectly regulate the phosphorylation of the retinoblastoma tumor suppressor gene product (pRB) and subsequent progression of the cell cycle. Inactivation of either TbetaR-II or TbetaR-I gene may lead to the deregulation of cell cycle and ultimately uncontrolled proliferation. The studies described herein address the hypothesis that a subset of the genetic changes commonly present in end-stage oral squamous cell carcinomas (SCC) will have already occurred in premalignant (leukoplakia, erythroplakia and proliferative verrucous leukoplakia) lesions. We have previously shown the TbetaR-II gene to be mutated (21%) and down-regulated (87%) in end-stage SCC of the head and neck. However, other potential mechanisms of this down- regulation, as well as a detailed evaluation of the TbetaR-I gene component in premalignant and malignant oral lesions have yet to be explored. Our experimental approach involves a combination of genetic and biochemical methodologies. Studies in Specific Aim 1 are designed to determine the incidence and mechanism of TbetaR-II and TbetaR-I gene inactivation (including deletions, insertions, single nucleotide substitutions, and hypermethylation events) in premalignant and malignant oral lesions. Once identified, oral lesions exhibiting TbetaR-II or TbetaR-I mutations will be assessed at the level transcription and/or translation for altered expression compared to patient-matched normal tissues (Specific Aim 2). These expression studies will be performed as a means of determining a precise level of TGF-beta receptor complex deregulation. In addition, specific TbetaR-II and TbetaR-I mutant proteins will be evaluated in Specific Aim 3 for their degree of biologic activity compared to the wild-type proteins. Mutant proteins will be constructed by site-directed mutagenesis and evaluated for their ligand binding, phosphorylation and trans-binding abilities in vitro. Overall, it is anticipated that the results from these studies will not only broaden our understand of the molecular mechanisms of oral cancer development, but may also aid in the identification of specific causative agents, intermediate endpoint biomarkers, and the development of successful intervention strategies which target premalignant disease.